Laser scanning optical system and laser scanning optical apparatus

ABSTRACT

A fluorescent microscope includes a laser scanning unit having a stage for supporting a fluorescent sample, a laser beam generator and a beam scanning unit for causing a laser beam from the generator to spot scan a sample to cause fluorescence generated by multi-photon absorption. A first detector detects fluorescence emitted from a side of the sample on which the laser beam is incident to output a first signal corresponding to detected fluorescence. A second detector detects fluorescence emitted from a side of the sample from which the laser beam, having been transmitted through the sample, is emitted from the sample. The second detector produces a second signal corresponding to the detected fluorescence. The first and second signals are summed and amplified. A display is provided for a microscopic image of the sample in synchronization with the scanning of the laser beam, based on the amplified summed signals.

This is a continuation of application Ser. No. 08/525,419, filed Sep. 7,1995, now U.S. Pat. No. 5,583,342, which is a continuation ofapplication Ser. No. 08/252,789, filed Jun. 2, 1994, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a laser scanning optical system and alaser scaning optical apparatus for performing scanning of laser beam,used in various fields, for example in biology, in medicine, insemiconductor engineering, as a fluorescence microscope, an opticalwriting apparatus, and as an IC repair apparatus.

2. Related Background Art

If an ordinary fluorescence microscope is used to observe a samplehaving a three-dimensional thickness, defocused images outside the depthof focus are superimposed on an image formed on the focal plane. Thisglobally lowers the contrast of microscopic image, which makesdetermination of fluorescence intensity difficult. Approaches to dealwith this problem will be described in the following.

For example, in a conventional confocal laser scanning fluorescencemicroscope, a laser beam emitted from a laser is expanded in diameter ofits ray bundle by a beam expander and thereafter passes through adichroic mirror. The laser oscillates the laser beam at a wavelengthcorresponding to a peak wavelength in an absorption spectrum of afluorescent dye used in labeling of a sample. The beam expander iscomposed of two convex lenses. The dichroic mirror is so formed as tohave a high reflectivity for optical components in a predeterminedwavelength range including a fluorescence emitted from the fluorescentdye and a high transmittance for the oscillation wavelength of the laserbeam.

The laser beam is bent in the direction perpendicular to the opticalaxis by an X-Y scanner and is focused by an imaging lens to form anoptical spot on a front image plane of an objective lens, and thereafterthe objective lens converges the optical spot to the diffraction limitto form a converged optical spot inside the sample. The X-Y scannerchanges the traveling direction of laser beam within a predeterminedangular range to scan in two orthogonal directions on a plane. Further,the objective lens or a stage on which the sample is set moves inparallel with the optical axis. Thus, the optical spot of the laser beamthree-dimensionally scans the inside of sample by parallel scanning tothe optical axis in addition to the two-dimensional scanningperpendicular to the optical axis, such as raster scanning. The sampleis, for example, an organism sample labeled with a fluorescent dye,which is excited by the optical spot of the laser beam.

A fluorescence diverging out of the sample is collected by the objectivelens and thereafter advances backward through the optical path, throughwhich the laser beam has passed. The fluorescence outgoing from the X-Yscanner is reflected by the dichroic mirror in the directionperpendicular to the optical axis and thereafter is focused by acollimator lens to form an image thereof in a pinhole in a confocalpinhole plate. The fluorescence outgoing from the confocal pinhole plateis separated from fluorescence components emitted from positions beforeand after the optical spot inside the sample and is received by a PMT(Photo Multiplier Tube).

The PMT photoelectrically converts the fluorescence into an electricsignal corresponding to the light intensity thereof and outputs it. Theelectric signal output from the PMT is stored as image data in a memoryin an image reading apparatus in synchronization with a scanning signalof the X-Y scanner. A three-dimensional microscopic image of the sampleis obtained by processing the image data in correspondence with thescanning signal by ordinary procedure.

The conventional confocal laser scanning fluorescence microscope asdescribed above employs such an arrangement that ideally a point lightsource and a point photodetector are located at positions conjugate witha point inside the sample whereby the laser beam forms an optical spothaving a reduced focal depth. Also, the pinhole is located on thephotodetector side so as to remove the fluorescence components emittedfrom positions before and after the optical spot inside the sample. Thiscan eliminate almost all defocused images except for an image on thefocal plane. Accordingly, only the image near the focal plane inside thesample is obtained as a microscopic image.

The prior art on such a confocal laser scanning fluorescence microscopeis described in detail, for example, in "Japanese Laid-open PatentApplication No. 2-247605".

Also, a conventional two-photon absorption excitation type laserscanning fluorescence microscope employs a laser oscillating a laserbeam as a pulse having a very short time duration, in which the laserbeam forms an optical spot having a high energy density and the opticalspot three-dimensionally scans the inside of a sample in the same manneras in the confocal laser scanning fluorescence microscope as describedpreviously. Because of the arrangement, a fluorescence due to excitationbased on two-photon absorption appears only from a point where theoptical spot is located inside the sample but no fluorescence due toexcitation based on two-photon absorption appears from other portions.Therefore, there appears no defocused image other than one on the focalplane, which improves the contrast of the microscopic image.

The prior art on such a two-photon absorption excitation type laserscanning fluorescence microscope is described in detail for example inreferences "Science, vol. 248, pp. 73-76, 6 Apr., 1990" and "U.S. Pat.No. 5,034,613, 1991".

Further, a conventional general laser scanning fluorescence microscopesemploy an axicon prism replacing the objective lens, by which laserbeams interfere with each other on the optical axis to be converted intoa bundle of rays having a focal depth as long as the thickness ofsample, i.e., into a so-called Bessel beam and through which the Besselbeam three-dimensionally scans the inside of sample in the same manneras in the confocal laser scanning fluorescence microscope as describedpreviously. Therefore, an image within the focal depth will never beunfocused, thus obtaining a microscopic image as a two-dimensionalprojection of a three-dimensional image. In another arrangement a laserbeam is guided through an aperture having an annular opening to beshaped in a cylindrical bundle of rays to enter an objective lens, thelaser beam forms a Bessel beam having a depth of focus as long as thethickness of the sample, and then the Bessel beam three-dimensionallyscans the inside of sample in the same manner as in the confocal laserscanning fluorescence microscope as described previously. Thus, an imagewithin the focal depth will never be unfocused, obtaining a microscopicimage as two-dimensional projection of a three-dimensional image.

A conventional optical converting unit for producing a Bessel beam is soarranged that an aperture having an annular opening portion is set onthe front focal plane of a convex lens. If a laser beam passes as a beamof parallel rays through the opening portion in the aperture, adiffracted light is produced as a bundle of rays having an annularcross-sectional intensity distribution perpendicular to the opticalaxis. The diffraction light having passed through the convex lensadvances as a plane wave refracted at a constant angle relative to theoptical axis and thereafter forms a conical wavefront in axial symmetrywith respect to the optical axis at the rear focus of the convex lens.Thus, beams of the diffracted light mutually interfere in the entireregion where the wavefront exists near the optical axis, so that aBessel beam is produced with an intensity enhanced by constructiveinterference.

In an intensity distribution of the Bessel beam in a cross sectionperpendicular to the optical axis, a thin linear center beam with strongintensity with the interference region near the optical axis existsalmost constantly. On the other hand, concentric cylindricalhigher-order diffraction beams with small intensity are present atpositions away from the optical axis. It is thus understood that theBessel beam has a high resolution and a long focal depth.

The prior art on such an annular illumination optical system using theaperture in the laser scanning fluorescence microscope is described indetail, for example, in "Optics, vol. 21, no. 7, pp. 489-497, July1992". Also, the prior art on production of the Bessel beam by theaxicon prism is described in detail, for example, in "Laser MicroscopeResearch Group, the tenth lecture papers, pp. 22-29, November 1992".Further, the prior art on the annular illumination optical system usingthe axicon prism is described in detail for example in "U.S. Pat. No.4,887,592, 1989".

However, the conventional confocal laser scanning fluorescencemicroscope as described above shields the fluorescence componentsemitted from positions before and after the optical spot inside thesample by locating a pinhole on the photodetector side. This extremelylowers the reception efficiency of fluorescence in the photodetector,which results in a problem of further reducing the intensity oforiginally weak fluorescence.

Also, in case of the two-photon absorption excitation type laserscanning fluorescence microscope as described above, the fluorescenceappears only from an optical spot inside the sample and therefore inorder to obtain a three-dimensional microscopic image, the optical spotis scanned in parallel with the optical axis inside the sample inaddition to the two-dimensional scanning perpendicular to the opticalaxis. This requires a long time as the scanning time of the opticalspot. In this time a sample of an organism having activity could move,resulting in failing to obtain a correct three-dimensional microscopicimage. In addition, there is a problem that a discoloration state or anexhaustion state of fluorescent dye becomes locally different.

Further, the conventional general laser scanning fluorescencemicroscopes as described above employ the axicon prism replacing theobjective lens whereby the laser beam forms a Bessel beam having a focaldepth as long as the thickness of sample. Thus, the axicon prism has noimaging function and a state of convergence is not good for the bundleof rays irradiated onto the sample, which results in a problem that theresolution is low in the plane perpendicular to the optical axis ofaxicon prism. In another case, a laser beam is guided through anaperture having an annular opening to form a bundle of rays having anannular cross section intensity perpendicular to the optical axis andthe bundle is made incident into the objective lens, whereby the laserbeam forms a Bessel beam having a focal depth as long as the thicknessof the sample. Even if the laser beam has a cross-sectional intensitydistribution based on an approximately Gaussian distribution, a bundleof rays with peak intensity is shielded by the disc shielding portion inthe aperture, which causes a problem that the utilization factor of thelaser beam irradiated onto the sample is extremely lowered. Also, thebundle of rays or the optical spot of the laser beam having a largefocal depth as described above forms annular beams of the higher-orderdiffraction beams around a linear beam. This causes a problem that whensuch a bundle of rays or the optical spot scans the inside of sample, alot of false signals are generated.

Therefore, the present invention has been accomplished in view of theabove problems and an object of the present invention is to provide alaser scanning optical system and a laser scanning optical apparatuswhich can perform scanning of laser beam with a higher energyutilization factor, a higher resolution and a longer focal depth withina shorter time than the prior art apparatus did.

SUMMARY OF THE INVENTION

A laser scanning optical system of the present invention, for achievingthe above object, comprises an optical converting unit for shaping alaser beam incident thereinto as a beam of parallel rays into acylindrical ray bundle, an optical scanning unit for changing atraveling direction of the laser beam incident from the opticalconverting unit thereinto to scan, and an optical converging unit forconverging the laser beam incident from the optical scanning unitthereinto to produce a Bessel beam, wherein the optical converting unitis composed of two axicon prisms which are arranged such that apexesthereof are opposed forward or backward to each other at a predetermineddistance and optical axes thereof are coincident with each other andwhich are made of respective materials having a same refractive indexand shaped with the same apical angle.

Here, the laser scanning optical system may have such a feature that theoptical converting unit further has moving means for variably setting adistance between the two axicon prisms.

Also, the laser scanning optical system may have such a feature that itfurther comprises a beam expander disposed either on the entrance sideor on the exit side of the optical converting unit, for expanding adiameter of the ray bundle of laser beam.

Further, the laser scanning optical system may have such a feature thatit also comprises a beam reducer disposed either on the entrance side oron the exit side of the optical converting unit, for reducing a diameterof the ray bundle of the laser beam.

Furthermore, a laser scanning optical apparatus of the presentinvention, for achieving the above object, comprises a light source foremitting a laser beam, an optical converting unit for shaping the laserbeam incident from the light source thereinto as a beam of parallel raysinto a cylindrical ray bundle thereof, an optical scanning unit forchanging a traveling direction of the laser beam incident from theoptical converting unit thereinto to scan, and an optical convergingunit for converging the laser beam incident from the optical scanningunit thereinto to irradiate a Bessel beam onto a predetermined sample,wherein the optical converting unit is composed of two axicon prismswhich are arranged such that apexes thereof are opposed forward orbackward to each other at a predetermined distance and optical axesthereof are coincident with each other and which are made of respectivematerials having a same refractive index and shaped with a same apicalangle.

Here, the laser scanning optical apparatus may have such a feature thatthe light source oscillates the laser beam as a pulse having a veryshort time duration, the sample is labeled with a predeterminedfluorescent dye, the apparatus further comprises an optical detectingunit for detecting fluorescence emitted from the sample based onmultiphoton absorption with irradiation of the Bessel beam, and outputsignals from the optical detecting unit are stored as pixel data insynchronization with scanning of the laser beam to obtain a microscopicimage of the sample.

In this case, it is preferred that the optical detecting unit comprisesa first photoelectric detector for detecting the fluorescence emittedfrom a surface side of the sample and a second photoelectric detectorfor detecting the fluorescence emitted from a back side of the sampleand that the microscopic image is produced based on addition of outputsignals from the first and second photoelectric detectors.

Also, the laser scanning optical apparatus may have such a feature thata surface of the sample is coated with a predetermined photosensitiveagent and a predetermined pattern is formed on the photosensitive agent,based on exposure with irradiation of the Bessel beam.

Further, the laser scanning optical apparatus may have such a featurethat a surface of the sample is exposed to the outside and a surfaceregion of the sample is shaped into a predetermined shape, based onexcitation with irradiation of the Bessel beam.

In the laser scanning optical system of the present invention, theoptical converting unit is composed of two axicon prisms. These axiconprisms are so arranged that their apexes are opposed forward or backwardto each other at a predetermined distance and their optical axes arecoincident with each other, and are made of respective materials havingthe same index of refraction and shaped with the same apical angle.

By this arrangement, the laser beam incident as a beam of parallel raysinto the optical converting unit is refracted at an equal angle relativeto the optical axis by one axicon prism to form a conical wavefront andthereafter becomes a divergent bundle of rays having an annularcross-sectional intensity perpendicular to the optical axis. A laserbeam emergent from this axicon prism is refracted at an equal anglerelative to the optical axis by the other axicon prism to become acylindrical bundle of rays as parallel beams with the travelingdirection being parallel to the optical axis.

The laser beam thus emerging from the optical converting unit is changedin the traveling direction by the optical scanning unit to scan and isconverged by the optical converging unit to become a Bessel beam. Adiffraction beam intensity distribution of the Bessel beam includes avery thin linear center beam having a strong intensity in the axialdirection and numerous concentric cylindrical higher-order diffractionbeams present around the center beam. Since the center beam has anextremely strong intensity as compared with those of the higher-orderdiffraction beams, the Bessel beam has a high resolution and a longfocal depth.

Accordingly, the laser scanning optical system of the present inventioncan form the cylindrical bundle of rays without losing the intensity ofthe laser beam, so that the scanning of Bessel beam can be carried outwith a high energy utilization factor.

For example, in case the optical converting unit further has the movingmeans for variably setting the distance between the two axicon prisms,the outer diameter of the cylindrical ray bundle outgoing from theoptical converting unit can be determined based on the distance betweenthe two axicon prisms. Since the annular zone width, which is adifference between the outer diameter and the inner diameter of thecylindrical ray bundle, is constant based on the diameter of ray bundleof the laser beam incident into the entrance-side axicon prism, a ratioof the inner diameter to the outer diameter of the cylindrical raybundle continuously changes according to the distance between the twoaxicon prisms. This changes the diameter and the intensity of the centerbeam relative to the higher-order diffraction beams in the Bessel beam,which makes adjustment of resolution and focal depth possible.

If there is a beam expander or a beam reducer further provided on theentrance side of the optical converting unit, the diameter of the raybundle of the laser beam incident on the optical converting unit can beadjustable. Then the annular zone width, which is a difference betweenthe outer diameter and the inner diameter of the cylindrical ray bundleoutgoing from the optical converting unit, is determined based on thediameter of ray bundle of the laser beam incident into the entrance-sideaxicon prism. Since the outer diameter of the cylindrical ray bundle isconstant based on the distance between the two axicon prisms, the ratioof the inner diameter to the outer diameter of the cylindrical raybundle continuously changes in correspondence with the beam expander orbeam reducer. Then the diameter and the intensity of the center beamchanges with respect to the higher-order diffraction beams in the Besselbeam, which makes adjustment of resolution and focal depth possible.

Also, if there is a beam expander or a beam reducer further provided onthe exit side of the optical converging unit, the outer diameter of acylindrical ray bundle outgoing from the optical converting unit is soadjusted as to coincide with the aperture diameter of the opticalconverging unit. This causes little eclipse of the laser beam. That is,the energy utilization factor of laser beam is increased, so that aBessel beam with high energy density can be produced.

In the laser scanning optical apparatus of the present invention a laserbeam emitted from a light source is irradiated onto a predeterminedsample through the laser scanning optical system of the presentinvention. By this arrangement, the sample is subjected to scanning witha Bessel beam having a high energy density, a high resolution and a longfocal depth.

For example, in case that the light source oscillates a laser beam ofvery-short-time-duration pulse, that the sample is labeled with apredetermined fluorescent dye, and that the apparatus further comprisesan optical detecting unit for detecting fluorescence emitted from thesample based on multiphoton absorption with irradiation of the Besselbeam, the optical detecting unit receives fluorescence from thefluorescent dye as excited at a wavelength corresponding to a fractionof the oscillating wavelength of the laser beam. The multiphotonabsorption occurs only in portions where the energy level of laser beamexceeds a predetermined value. Then, if the output of the laser beam isadjusted such that the multiphoton absorption is not caused by thehigher-order diffraction beams accompanying the center beam in theBessel beam, fluorescence will appear only from a position irradiated bythe center beam in the Bessel beam within the sample. This can preventfalse signals from being produced by the accompanying higher-orderdiffraction beams in the Bessel beam, which can enhance the resolutionof a microscopic image of the sample as obtained by storing as pixeldata output signals from the optical detecting unit in synchronizationwith the scanning with the laser beam.

Two-dimensional scanning is carried out inside the sample with theBessel beam having the linearly formed center beam, so that amicroscopic image is obtained as two-dimensional projection of athree-dimensional image of the sample in the form of integral values inthe thickness direction. This can obviate three-dimensional scanninginside the sample, which can greatly reduce the scanning time of opticalspot.

Further, if the optical detecting unit is composed of a firstphotoelectric detector for detecting fluorescence emitted from thesurface side of the sample and a second photoelectric detector fordetecting fluorescence emitted from the back side of the sample, thefluorescence emitted from the sample can reach the respectivephotoelectric detectors without any loss. This can improve the contrastin the microscopic image as produced based on addition of output signalsfrom the photoelectric detectors. Therefore, a low-power laser can beused as the light source for emitting the laser beam, which can reducedamage to an organism sample or other type of sample.

In case the surface of sample is coated with a predeterminedphotosensitive agent and a selected pattern is formed on thephotosensitive agent, based on exposure with irradiation of the Besselbeam, exposure can be well done for the photosensitive agent on thesubstrate irrespective of unevenness of the surface of substrate. Thiscan obviate precise alignment of the position of optical spot of thelaser beam with respect to the photosensitive agent, improving theefficiency of operation. Since the Bessel beam has a high resolution, itcan be applied to exposure of an integrated circuit pattern on aphotosensitive agent on a substrate, whereby the degree of integrationcan be increased for integrated circuits formed based on this pattern.

Further, in case the surface of sample is exposed to the outside and asurface region of sample is shaped in a predetermined shape based onexcitation with irradiation of the Bessel beam, the surface region ofthe substrate can be etched without being affected by unevenness ofitself, because the Bessel beam has a long focal depth. This can obviateprecise alignment of the position of optical spot of laser beam withrespect to the substrate, which improves the efficiency of operation.Since the Bessel beam has a high resolution and if the sample is an ICchip having integrated circuits on its surface region, a structurallyfine defect occurring in the integrated circuits can be repaired.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a structural drawing to show a first embodiment associatedwith a fluorescence microscope using a laser scanning optical system ofthe present invention.

FIG. 2 is a structural drawing to show an axicon pair in thefluorescence microscope of FIG. 1 in more detail.

FIG. 3 is a perspective view to show the structure of axicon prisms inthe axicon pair of FIG. 2.

FIG. 4 is composed of a graph to show an intensity distribution in aplane perpendicular to the direction of the optical axis as to a laserbeam incident into the axicon pair of FIG. 2, a structural drawing toshow a case in which a distance between the two axicon prisms in theaxicon pair of FIG. 2 is variably set, and a graph to show an intensitydistribution in a plane perpendicular to the direction of the opticalaxis as to a laser beam outgoing from the axicon pair of FIG. 2.

FIG. 5 is a graph to show a change of intensity distribution of Besselbeam in a plane perpendicular to the optical axis with a change of theratio of the inner diameter to the outer diameter of the cylindrical raybundle.

FIG. 6 is a graph to show an enlarged main portion in the intensitydistribution of FIG. 5.

FIG. 7 is a structural drawing to show a second embodiment associatedwith a fluorescence microscope using a laser scanning optical system ofthe present invention.

FIG. 8 is a perspective view to show the structure of a laminated sensorarray in the fluorescence microscope of FIG. 7.

FIG. 9 is a cross sectional view along the lamination thicknessdirection of a single sensor in the laminated sensor array of FIG. 8.

FIG. 10 is a structural drawing to show a third embodiment associatedwith a fluorescence microscope using a laser scanning optical system ofthe present invention.

FIG. 11 is a structural drawing to show an embodiment associated with anoptical writing apparatus using a laser scanning optical system of thepresent invention.

FIG. 12 is a structural drawing to show an embodiment associated with alaser repair apparatus using a laser scanning optical system of thepresent invention.

FIGS. 13(a)-13(c) are structural drawings to show a beam expander or abeam reducer in the fluorescence microscope of FIG. 1 in more detail.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The structure and operation of embodiments according to the presentinvention will be described with reference from FIG. 1 to FIG. 12. Inthe description of the drawings the same elements will be denoted bysame reference numerals and redundant description will be omitted. Itshould be also noted that the dimensions of the drawings do not alwayscoincide with those in the description.

FIG. 1 is a structural drawing showing a first embodiment associatedwith a fluorescence microscope using the laser scanning optical systemof the present invention. In the fluorescence microscope, a plane mirror3 is disposed along a traveling direction of a laser beam 2 emitted froma laser (light source) 1. Along the traveling direction of laser beam 2reflected by the plane mirror 3 in the direction perpendicular to theoptical axis, there is a beam expander 4, an axicon pair (opticalconverting unit) 6, a beam reducer 9, a dichroic mirror 11a and an X-Yscanner (optical scanning unit) 12 arranged approximately on a line.Along the traveling direction of the laser beam 2 outgoing from the X-Yscanner 12 in the direction perpendicular to the optical axis, there isan imaging lens 13, an objective lens 14 (optical converging unit) and asample 15 arranged approximately on a line. Along the travelingdirection of fluorescence 16b emitted from the front side of the sample15, there is a condenser lens 17, a barrier filter 18 and a PMT 19aarranged approximately on a line. On the other hand, there is a PMT 20aarranged along the traveling direction of fluorescence 16a emitted fromthe back side of the sample 15 and then passing through the objectivelens 14, the imaging lens 13 and the X-Y scanner 12 to be furtherreflected by the dichroic mirror 11a in the direction perpendicular tothe optical axis.

The laser 1 is a light source for oscillating the laser beam 2 in apulse having a very short time duration. The laser beam 2 is a beam ofparallel rays having an optical pulse duration of several ten to severalhundred fs, a repetitive frequency of several ten to several hundred MHzand a wavelength two or three times longer than the peak wavelength inan absorption spectrum of a fluorescent dye for labeling the sample 15.The plane mirror 3 has a mirror surface inclined approximately at 45degrees relative to the traveling direction of laser beam 2 emitted fromthe laser 1 and the mirror surface reflects the laser beam 2 incidentthereinto from the laser 1.

The beam expander 4 is composed of two convex lenses 5a, 5b, whichenlarges the ray bundle of the laser beam 2 incident thereon from theplane mirror 3 into a bundle having a predetermined diameter. Theseconvex lenses 5a, 5b are so arranged at a predetermined distance thatthe optical axes thereof are coincident with each other.

It is also preferred that the beam expander 4 is further composed aconcave lens 5c arranged between two convex lenses 5a, 5b as shown inFIG. 13(a) to variably set the distance between the convex lens 5a andthe concave lens 5c by unrepresented moving means of the concave lens5c. In this arrangement, the convex lenses 5a, 5b and the concave lens5c respectively function as a focus-lag-correction lens group, a fixedlens group and a zoom lens group. Therefore a magnification ratio forthe diameter of the ray bundle of laser beam 2 is continuouslychangeable or zoomed when the position of the concave lens 5c is changedfrom a region as shown in FIG. 13(b) to the region shown FIG. 13(a).

FIG. 2 is a structural drawing to show the axicon pair 6 in more detail.FIG. 3 is a perspective view to show the configuration of an axiconprism in the axicon pair 6. The axicon pair 6 is composed of two axiconprisms 7a, 7b, a motor 7c, a rotation axis 7d, a pinion gear 7e and arack gear 7f. These axicon prisms 7a, 7b are made of respectivematerials having the same index of refraction in the shape of a circularcone. Also, the axicon prisms 7a, 7b are so arranged that the apicalangles thereof having the same angle are opposed forward or backward toeach other at a predetermined distance and the optical axes thereof arecoincident with each other.

A distance between such axicon prisms 7a, 7b is variably set byunrepresented moving means. For example, in such a moving means thepinion gear 7c is set at the distal portion of the rotation axis 7dwhich transmits the drive of the motor 7c, and the rack gear 7e is setat the distal portion of the axicon prism 7b. The gear teeth of thepinion gear 7e and the rack gear 7f are so combined that the rotationdirection of the pinion gear 7e and the moving direction of the rackgear 7f are arranged along a direction parallel to the optical axis. Inthis arrangement, when the motor 7c rotates in a predetermineddirection, the axicon prism 7b moves in the direction corresponding tothe rotation direction of the motor 7c along the optical axis. Thereforethe distance between the axicon prisms 7a, 7b changes relatively. Here,it is also preferred that the structure of such a rack and pinion isrespectively set at both of the axicon prism 7a, 7b and is set at leastat one or the other.

FIG. 4 is a graph showing an intensity distribution in a planeperpendicular to the optical axis as to the laser beam 2 incident intothe axicon pair 6, a structural drawing showing a case in which thedistance between the two axicon prisms 7a, 7b is variably set, and agraph to show an intensity distribution in a plane perpendicular to theoptical axis as to the laser beam 2 outgoing from the axicon pair 6.

The axicon pair 6 is so arranged that the laser beam 2 is incident as abeam of parallel rays having cross-sectional intensities based on asubstantial Gaussian distribution perpendicular to the optical axis,from the beam expander 4 into the axicon prism 7a, and that the axiconprism 7a refracts the laser beam 2 at a constant angle relative to theoptical axis to emit a conical ray bundle having an annularcross-sectional intensity perpendicular to the optical axis. On theother hand, the axicon prism 7b refracts the laser beam 2 incidentthereon from the axicon prism 7a at a constant angle relative to theoptical axis, to emit a cylindrical ray bundle 8 traveling in thedirection parallel to the optical axis. The outer diameter α of thecylindrical ray bundle 8 is determined by the distance between theaxicon prisms 7a, 7b, and the annular zone width, which is a differencebetween the outer diameter α and the inner diameter α', is determinedbased on the diameter of the ray bundle of the laser beam 2 incident onthe axicon prism 7a. Therefore, a ratio β (=α'/α) of the inner diameterα' to the outer diameter α can be set while continuously changed orzoomed in correspondence with the distance between the axicon prisms 7a,7b.

The beam reducer 9 is composed of two convex lenses 10a, 10b, whichreduces the outer diameter of the laser beam 2 incident as thecylindrical ray bundle 8 thereon from the axicon pair 6 so as to matchwith the aperture diameter of the objective lens 14. These convex lenses10a, 10b are so arranged at a predetermined distance that the opticalaxes thereof are coincident with each other.

It is also preferred that the beam reducer 9 is further composed of aconcave lens 10c arranged between two convex lenses 10a, 10b as shown ina FIG. 13(c) to variably set the distance between the convex lens 10aand the concave lens 10c by unrepresented moving means of the concavelens 10c. In this arrangement, the convex lenses 10a, 10b and theconcave lens 10c respectively function as a focus-lag-correction lensgroup, a fixed lens group and a zoom lens group. Therefore amagnification ratio for the diameter of ray bundle of laser beam 2 iscontinuously changeable or zoomed when the postion of the concave lens10c is changed from a FIG. 13(b) to the FIG. 13(c).

The dichroic mirror 11a is so formed as to have a large transmittancefor the oscillation wavelength of the laser beam 2 incident from thebeam reducer 9 and a large reflectivity for light components in apredetermined wavelength range including the fluorescence 16a incidentfrom the X-Y scanner 12. The X-Y scanner 12 is for example agalvanometer scanner, a resonant scanner, a piezo oscillation scanner, arotary polygon scanner, an ultrasonic vibrator deflector (Acousto-OpticDeflector), etc. The X-Y scanner 12 changes the traveling direction ofthe laser beam 2 incident thereon from the dichroic mirror 11a within apredetermined angular range to scan the beam in two orthogonaldirections in a plane and similarly changes the traveling direction offluorescence 16a incident thereon from the imaging lens 13 to deflect ittoward the dichroic mirror 11a.

The imaging lens 13 focuses the laser beam 2 incident thereon from theX-Y scanner 12 to form a beam spot on the front image plane of theobjective lens 14. The fluorescence 16a is focused as a beam spot on therear image plane of the imaging lens 13 and the fluorescence 16aincident from the beam spot as a virtual light source into the imaginglens 13 is emergent as a beam of parallel rays therefrom. The objectivelens 14 converges the laser beam 2 incident as a virtual light source ofa beam spot formed on the front image plane thereof to produce a Besselbeam as a beam spot converged up to the diffraction limit and to let theBessel beam pass through the inside of sample. The objective lens 14collects the fluorescence 16a emitted from the surface side of sample 15to form a beam spot on the rear image plane of imaging lens 13.

Here, near the rear focus of objective lens 14 the shape ofthree-dimensional intensity distribution of the Bessel beam changesdepending upon the annular zone width of the cylindrical ray bundle 8.That is, the Bessel beam changes the intensity distribution of thecenter beam and higher-order diffraction beams in accordance with achange of ratio β (=α'/α) of the inner diameter α' to the outer diameterα of the cylindrical ray bundle 8.

FIG. 5 is a graph showing a change of intensity distribution of Besselbeam in a plane perpendicular to the optical axis with a change of theratio of the inner diameter to the outer diameter of the cylindrical raybundle 8. FIG. 6 is a graph showing an enlarged main portion in theintensity distribution of FIG. 5. Here, the scale unit on the horizontalaxis is shown as 2pNA/λ (NA: the numerical aperture of the objectivelens, λ: the wavelength of the laser beam). The scale on the verticalaxis is shown as normalized based on the intensity in correspondencewith the peak wavelength of the laser beam.

As the ratio β of the inner diameter to the outer diameter of thecylindrical ray bundle 8 approaches 1, the spot diameter of the centerbeam decreases while the intensity of higher-order diffraction beamsincreases relative to the center beam. Also, the shape of the centerbeam and higher-order diffraction beams becomes longer in the directionof the optical axis. Consequently, it is seen that if the shape of thecylindrical ray bundle 8 is annular with a narrower width, the Besselbeam increases the focal depth and the resolution.

The sample 15 is for example an organism sample labeled with apredetermined fluorescent dye, which is located nearly at a positionwhich is at the rear image plane of objective lens 14 and the frontimage plane of condenser lens 17. The sample 15 is excited to emitfluorescence 16a, 16b, based on the multiphoton absorption at theposition irradiated by the laser beam 2 incident as the Bessel beam fromthe objective lens 14. Also, the sample 15 is set on an unrepresentedstage and is moved along the optical axis together with the stage.

The condenser lens 17 collects the laser beam 2 and the fluorescence 16boutgoing from the back side of sample 15 to emit them as parallel rays.The barrier filter 18 has a high transmittance for light components in apredetermined wavelength range including the fluorescence 16b emergentfrom the sample 15 and a large absorbance for the oscillation wavelengthof the laser beam 2 emergent from the sample 15.

The PMTs (Photo-Multiplier Tube) 19a, 20a are photoelectric detectorssuch as photomultipliers. The PMT 19a receives the fluorescence 16boutgoing from the barrier filter 18 and photoelectrically converts itinto an electric signal reflecting the optical intensity thereof tooutput the electric signal. On the other hand, the PMT 20a receives thefluorescence 16a incident thereon from the dichroic mirror 11a andphotoelectrically converts it into an electric signal reflecting theoptical intensity thereof to output the electric signal.

Next described is the operation of the above first embodiment associatedwith the fluorescence microscope.

The laser beam 2 represented by the solid line is emitted from the laser1 and thereafter is reflected by the plane mirror 3 in the directionperpendicular to the optical axis. The beam expander 4 expands thediameter of ray bundle of laser beam 2 coming from the plane mirror 3into a predetermined value while keeping the traveling direction thereofaligned with the direction of the optical axis.

In the axicon pair 6, all the laser beam 2 outgoing as collimated planewaves from the beam expander 4 is refracted by the axicon prism 7a atthe same angle relative to the optical axis to form a conical wavefrontand thereafter to become a diverging bundle of rays having an annularcross-sectional intensity perpendicular to the optical axis. All oflaser beam 2 outgoing from the axicon prism 7a is refracted by theaxicon prism 7b at the same angle relative to the optical axis to becomea cylindrical ray bundle 8 in the form of a beam of parallel raystraveling in the direction parallel to the optical axis. Here, the beamdiameter of the cylindrical ray bundle 8 shown by the solid lines isfreely and continuously variable according to the distance between theaxicon prisms 7a, 7b, and the beam diameter is so set as to coincidewith the aperture diameter of the objective lens 14 under the overallmagnification including the pupil image magnification of the imaginglens 13 after having passed through the beam reducer 9.

The beam reducer 9 reduces the diameter of the ray bundle of the laserbeam 2 outgoing as the cylindrical ray bundle 8 from the axicon pair 6into a predetermined value while keeping the traveling direction thereofaligned with the direction of the optical axis. On this occasion theannular zone width is simultaneously compressed in the annular crosssection perpendicular to the optical axis in the cylindrical ray bundle8. The laser beam 2 outgoing from the beam reducer 9 passes through thedichroic mirror 11a and then is made outgoing in the directionperpendicular to the optical axis by the X-Y scanner 12. The X-Y scanner12 changes the traveling direction of the laser beam 2 within apredetermined angular range to deflect it in two orthogonal directionsin a plane.

The laser beam 2 outgoing from the X-Y scanner 12 is focused by theimaging lens 13 to form a beam spot on the front image plane ofobjective lens 14. It can be said that the beam spot is a virtual lightsource as two-dimensionally deviated by the X-Y scanner 12. The laserbeam 2 outgoing as the conical ray bundle from the beam spot passes onlythrough the peripheral portion of the objective lens 14 to be outputtherefrom and is allowed to impinge on the sample 15 as the Bessel beamwhich is a beam spot converged up to the diffraction limit. The Besselbeam has a diffraction beam intensity distribution composed of a verythin linear center beam having a large intensity in one direction of theoptical axis and numerous concentric cylindrical higher-orderdiffraction beams existing around the center beam. Also, the X-Y scanner12 makes the Bessel beam effect the two-dimensional scanning, forexample the raster scanning, inside the sample 15.

Inside the sample 15 the so-called multiphoton absorption occurs only ina portion irradiated by the Bessel beam having an optical intensity partexceeding a predetermined threshold. By this, the labeling fluorescentdye in the sample 15 emits the fluorescence 16a, 16b represented by thebroken lines as a linear, secondary light source proportional to anamount of the fluorescent dye similarly as in case of excitation by apeak wavelength in the absorption spectrum corresponding to a half orone third of the oscillation wavelength of laser beam 2.

The laser beam 2 passing through and outgoing from the sample 15 passesthrough the condenser lens 17 to become a beam of parallel raystraveling in parallel with the optical axis and thereafter to beabsorbed by the barrier filter 18. The fluorescence 16b outgoing fromthe back side of the sample 15 passes through the condenser lens 17 tobecome a beam of parallel rays traveling in parallel with the opticalaxis and thereafter passes through the barrier filter 18 then to bereceived by the PMT 19a. On the other hand, the fluorescence 16aoutgoing from the surface side of sample 15 travels backward in theoptical path through which the laser beam 2 has passed while condensedby the objective lens 14. The fluorescence 16a outgoing from the X-Yscanner 12 is reflected by the dichroic mirror 11a in the directionperpendicular to the optical axis and thereafter is received by the PMT20a.

The PMTs 19a, 20a output electric signals corresponding to the opticalintensities of fluorescence 16a and fluorescence 16b, respectively,after photoelectric conversion, and the electric signals are added toeach other. The signal thus added is stored as pixel data in a memory inan unrepresented image reading apparatus in synchronization with ascanning signal of the X-Y scanner 12. Processing this pixel data incorrespondence with the scanning signal by ordinary procedure, amicroscopic image is obtained as two-dimensional projection of athree-dimensional image of the sample 15.

With the first embodiment associated with the fluorescence microscope asdescribed above, the laser beam oscillated as a very short time durationpulse is converted using the two axicon prisms into the cylindrical raybundle having the narrow, annular cross-sectional intensityperpendicular to the optical axis. By this, there is almost no loss inintensity of laser beam caused in producing the cylindrical ray bundle.This can increase the energy utilization factor of laser beam and canmaintain high the energy density, which contributes to the multiphotonabsorption inside the sample.

Also, the laser beam oscillated as the very short time duration pulsepasses only through the peripheral portion of objective lens in the formof the cylindrical ray bundle, whereby the Bessel beam with the thinlinear center beam formed with a large intensity in the direction of theoptical axis is irradiated into the sample. This excites the sample onlyat the position irradiated by the Bessel beam by the multiphotonabsorption. Then, the fluorescence emitted based on the labelingfluorescent dye in the sample is received with high efficiency by thephotoelectric detectors without a need to use a pinhole. Also, themultiphoton absorption in the sample can be made to occur only in a verythin portion further closer to the center region of the Bessel beam byadjusting the energy density of laser beam. Then the fluorescencemicroscope may have a further higher resolution and a longer focaldepth.

In addition, the fluorescence emitted from both the surface and backsides of the sample is received by a plurality of photoelectricdetectors and a microscopic image is obtained by adding the outputsignals from these photoelectric detectors. This permits thefluorescence to reach the photoelectric detectors with almost no loss.Therefore, a low-power laser can be employed, which can reduce damage toan organism used as a sample or another sample.

Further, the Bessel beam has the linearly formed center beam, with whichthe two-dimensional scanning is performed inside the sample. Thisprovides a microscopic image in which a three-dimensional image ofsample is two-dimensionally projected, as integration values in thethickness direction. This can obviate the three-dimensional scanninginside the sample, which greatly decreases the scanning time of opticalspot.

FIG. 7 is a structural drawing to show a second embodiment associatedwith the fluorescence microscope using the laser scanning optical systemof the present invention. The present embodiment is constructed almostin the same manner as the above first embodiment associated with thefluorescence microscope. However, the present embodiment excludes thecondenser lens 17, the barrier filter 18 and the PMT 19a for measuringthe fluorescence emitted from the back side of sample 15. Also, on thereflection side of the dichroic mirror 11a there are a collimator lens21 and a laminated sensor array 22 arranged approximately on a linealong the traveling direction of fluorescence 16a emitted from thesurface side of sample 15. The collimator lens 21 is so arranged thatthe rear focus thereof is located at the center of the laminated sensorarray 22. Also, the laminated sensor array 22 is so arranged that thecenter axis in the lamination direction thereof is coincident with alinear fluorescent image of fluorescence 16a.

FIG. 8 is a perspective view to show the structure of the laminatedsensor array 22. FIG. 9 is a cross sectional view of a sensor in thelaminated sensor array 22 along the lamination thickness direction. Thelaminated sensor array 22 is composed of numerous photoelectric sensorsconcentrically laminated. Each photoelectric sensor is so constructedthat a photoelectric detecting portion having a p-type Si layer 25 offlat circular cylinder on an n-type Si layer 24 of circular cylinder isformed on a transparent substrate 23. Transparent electrodes 27 foroutputting signal charge are provided through insulating layers of SiO₂layers 26, 28 on the transparent substrate 23 including thephotoelectric detecting portion. Metal lines 29 are connected to thetransparent electrodes 27, and are exposed to the outside.

Next described is the operation of the above second embodimentassociated with the fluorescence microscope.

The present embodiment is operated almost in the same manner as theabove first embodiment associated with the fluorescence microscope.However, the fluorescence 16a emitted from the surface side of sample 15is collected by the objective lens 14, travels backward in the opticalpath where the laser beam 2 has passed, and is reflected by the dichroicmirror 11a in the direction perpendicular to the optical axis. Afterthat, it passes through the collimator lens 21 to form a beam spot atthe center of the laminated sensor array 22. The optical spot produces athin linear fluorescence image near the rear focus of collimator lens21, which is received by the laminated sensor array 22. The laminatedsensor array 22 converts the fluorescence 16a into an electric signal bythe laminated photoelectric sensors and outputs the electric signalthrough the metal lines 29.

Electric signals output from the photoelectric sensors in the sensorarray 22 by photoelectric conversion in correspondence with the opticalintensity are stored in parallel as pixel data in a memory in anunrepresented image reading apparatus in synchronization with thescanning signal of the X-Y scanner 12. The pixel data is processed incorrespondence with the scanning signal by ordinary procedure, so thatmicroscopic images are simultaneously obtained as two-dimensionalcross-sectional images obtained by dividing a three-dimensional image ofthe sample 15 into pieces in the number of photoelectric sensors.

With the above second embodiment associated with the fluorescencemicroscope, the Bessel beam having the linearly formed center beam isused to perform the two-dimensional scanning inside the sample and thefluorescence diverging from the sample is focused by the collimator lensto form a thin linear optical spot in the laminated sensor array to bereceived thereby. By this, a three-dimensional image of the sample isobtained as a lot of two-dimensional cross-sectional images divided inthe direction of the optical axis. This can obviate necessity ofthree-dimensional scanning inside the sample, which greatly reduces thescanning time of optical spot.

FIG. 10 is a structural drawing to show a third embodiment associatedwith the fluorescence microscope using the laser scanning optical systemof the present invention. The present embodiment is constructed almostin the same manner as the above first embodiment associated with thefluorescence microscope. However, the sample 15 is labeled with pluraltypes of fluorescent dyes and the fluorescent dyes have mutuallydifferent fluorescence emission wavelengths. Also, on the reflectionside of a dichroic mirror 11a there are dichroic mirrors 11b, 11c and aPMT 20a arranged approximately on a line along the traveling directionof fluorescence 16a emitted from the surface side of sample 15. PMTs20b, 20c are disposed on the reflection side of the dichroic mirrors11b, 11c, respectively. On the other hand, on the exit side of barrierfilter 18 there are dichroic mirrors 31b, 31c and a PMT 19a arrangedapproximately on a line along the traveling direction of fluorescence16b emitted from the back side of sample 15. PMTs 19b, 19c are disposedon the reflection side of the dichroic mirrors 31b, 31c, respectively.

The dichroic mirror 11a is so formed as to have a large transmittancefor the oscillation frequency of laser beam 2 incident thereinto fromthe beam reducer 9 and a large reflectivity for light components in apredetermined wavelength range including the fluorescence 16a incidentthereinto from the X-Y scanner 12. The dichroic mirror 11b, 11c has alarge reflectivity for either of mutually different wavelength bandsincluding associated fluorescence components with mutually differentemission wavelengths present in the fluorescence 16a emitted from thedichroic mirror 11a. The dichroic mirror 31b, 31c has a largereflectivity for either of mutually different wavelength bands includingassociated light components with mutually different emission wavelengthspresent in the fluorescence 16b outgoing from the barrier filter 18.

The PMTs 19a-19c, 20a-20c are photoelectric detectors such asphotomultipliers. The PMT 19b receives the fluorescence 16b reflected bythe dichroic mirror 31b and photoelectrically converts it into anelectric signal corresponding to the optical intensity to output theelectric signal. The PMT 19c receives the fluorescence 16b reflected bythe dichroic mirror 31c and photoelectrically converts it into anelectric signal corresponding to the optical intensity to output theelectric signal. The PMT 19a receives the fluorescence 16b passingthrough the dichroic mirrors 31b, 31c and photoelectrically converts itinto an electric signal corresponding to the optical intensity to outputthe electric signal. The PMT 20b receives the fluorescence 16a reflectedby the dichroic mirror 11b and photoelectrically converts it into anelectric signal corresponding to the optical intensity to output theelectric signal. The PMT 20c receives the fluorescence 16a reflected bythe dichroic mirror 11c and photoelectrically converts it into anelectric signal corresponding to the optical intensity to output theelectric signal. The PMT 20a receives the fluorescence 16a passingthrough the dichroic mirrors 11b, 11c and photoelectrically converts itinto an electric signal corresponding to the optical intensity to outputthe electric signal.

Next described is the operation of the third embodiment associated withthe fluorescence microscope.

The present embodiment is operated almost in the same manner as theabove first embodiment associated with the fluorescence microscope.However, the fluorescence 16a emitted from the surface side of sample 15is collected by the objective lens 14 to travel backward in the opticalpath through which the laser beam 2 has passed, it is then reflected bythe dichroic mirror 11a in the direction perpendicular to the opticalaxis, some light components are reflected by the dichroic mirrors 11b,11c to be received by the PMTs 20b, 20c, respectively and the otherlight components are received by the PMT 20a.

On the other hand, the fluorescence 16b emitted from the back side ofsample 15 is collected by the condenser lens 17 to become a beam ofparallel rays traveling in the traveling direction, the beam passesthrough the barrier filter 18, thereafter some light components arereflected by the dichroic mirrors 31b, 31c to be received by the PMTs19b, 19c, respectively, and the other light components are received bythe PMT 19a.

Added to each other are an electric signal output from each PMT 20a-20cafter photoelectric conversion corresponding to the optical intensity ofan associated light component in the fluorescence 16a and an electricsignal output from corresponding PMT 19a-19c after photoelectricconversion corresponding to the optical intensity of an associated lightcomponent in the fluorescence 16b. The electric signals as so added arestored as pixel data in a memory in an unrepresented image readingapparatus in synchronization with scanning signals of the X-Y scanner12. Processing the pixel data in correspondence with the scanningsignals based on ordinary procedure, a microscopic image astwo-dimensional projection of a three-dimensional image of sample 15 isobtained for each fluorescence component in the fluorescence 16a, 16b,i.e., for each emission wavelength of labeling fluorescent dye in thesample 15.

In the third embodiment associated with the fluorescence microscope asdescribed above, the sample is labeled with plural types of fluorescentdyes having mutually different emission wavelengths, and lightcomponents in the fluorescence diverging from the sample are separatedfrom each other by the dichroic mirrors to be received by thephotoelectric detectors. By this arrangement, if the two-dimensionalscanning is once executed inside the sample with the Bessel beam havingthe linearly formed center beam, a three-dimensional image of the sampleis formed as a plurality of microscopic images simultaneously obtainedas two-dimensional projection based on the emission wavelengths of thefluorescent dyes. This can obviate three-dimensional scanning inside thesample, which can greatly reduce the scanning time of the optical spot.

FIG. 11 is a structural drawing to show an embodiment concerning anoptical writing apparatus using the laser scanning optical system of thepresent invention. In the optical writing apparatus there are an opticalmodulator 32 and a plane mirror 3 arranged along the traveling directionof laser beam 2 emitted from a laser 1 (light source). Along thetraveling direction of the laser beam 2 reflected by the plane mirror 3in the direction perpendicular to the optical axis there is an axiconpair (optical converting unit) 6, a beam reducer 33 and an X-Y scanner(optical scanning unit) 12 arranged approximately on a line. Along thetraveling direction of the laser beam 2 outgoing from the X-Y scanner 12in the direction perpendicular to the optical axis, there is an imaginglens 13, an aperture stop 35, an objective lens 14 (optical convergingunit) and an X-Y stage 36 arranged approximately on a line. Inputterminals of the optical modulator 32, the X-Y scanner 12 and the X-Ystage 36 are electrically connected to associated output terminals of acontrol unit 37. A substrate 38 coated with a photosensitive agent 39 isset on the X-Y stage 36.

The optical modulator 32 is an optical modulator for modulating theintensity, the frequency, the phase and the plane of polarization of thelaser beam 2 incident thereon from the laser 1. Particularly, it selectseither one of continuous irradiation and pulse irradiation of the laserbeam 2 by the intensity modulation. The beam reducer 33 is composed of aconvex lens 34a and a concave lens 34b, which reduces the outer diameterof the laser beam 2 incident as a cylindrical ray bundle 8 thereon fromthe axicon pair 6 so as to make it coincident with the aperture diameterof objective lens 14. The convex lens 34a and concave lens 34b are soarranged that the optical axes thereof are coincident with each other.It is also preferred that the beam reducer 33 is further composed aconcave lens arranged between two convex lenses 34a, 34b as the beamreducer 9 in FIG. 13(c), to variably set the distance between the convexlens 34a and the concave lens by unrepresented moving means of theconcave lens. In this arrangement, the convex lenses 34a, 34b and theconcave lens respectively function as a focus-lag-correction lens group,a fixed lens group and a zoom lens group. Therefore a magnificationratio for the diameter of ray bundle of laser beam 2 is continuouslychangeable or zoomed when the postion of the concave lens is changedFIG. 13(b) to FIG. 13(c).

The aperture stop 35 is an aperture having a circular opening, whichlimits the outer diameter of ray bundle of the laser beam 2 incident asthe cylindrical ray bundle 8 thereinto from the imaging lens 13. The X-Ystage 36 moves the substrate 38 in a direction conjugate with thescanning direction of laser beam 2 by the X-Y scanner 12. The controlunit 37 is a microcomputer or the like, which sets optical modulation ofthe laser beam 2 through the optical modulator 32 and which setsscanning speeds and scanning timings of X-Y scanner 12 and X-Y stage 36in synchronization. The substrate 38 is a wafer made of a semiconductormaterial or a chip separated out of the wafer, and is located on therear image plane of objective lens 14. The photosensitive agent 39 is aphotoresist or the like, which is subjected to exposure with a highcontrast at the position irradiated by the laser beam 2 incident as aBessel beam thereon from the objective lens 14.

Next described is the operation of the above embodiment concerning theoptical writing apparatus.

The laser beam 2 emitted from the laser 1 is optically modulated by theoptical modulator 32 and thereafter is reflected by the plane mirror inthe direction perpendicular to the optical axis. In the axicon pair 6the axicon lenses 7a, 7b change the laser beam 2 outgoing from the planemirror 3 into a beam of parallel rays traveling in parallel with theoptical axis, which is a cylindrical ray bundle 8 having an annularcross-sectional intensity perpendicular to the optical axis. Here, thediameter of the cylindrical ray bundle 8 shown by the solid lines can befreely and continuously changed in correspondence with a distancebetween the axicon prisms 7a, 7b and is so set as to coincide with theaperture diameter of objective lens 14 under an overall magnificationincluding the pupil imaging magnification of imaging lens 13 after thebeam has passed through the beam reducer 33.

The beam reducer 33 reduces the diameter of ray bundle of the laser beam2 outgoing as the cylindrical ray bundle 8 from the axicon pair 6 into apredetermined value while maintaining the traveling direction thereofaligned with the direction of the optical axis. On this occasion theannular zone width is simultaneously compressed in annular cross sectionperpendicular to the optical axis in the cylindrical ray bundle 8. TheX-Y scanner 12 makes the laser beam 2 outgoing from the beam reducer 33directed in the direction perpendicular to the optical axis and changesthe traveling direction of the beam within a predetermined angular rangeto scan in two orthogonal directions in a plane.

The laser beam 2 outgoing from the X-Y scanner 12 is focused by theimaging lens 13 to form an optical spot on the front image plane ofobjective lens 14. The optical spot can be said as a virtual lightsource which can be two-dimensionally deviated by the X-Y scanner 12.For the laser beam 2 outgoing as the cylindrical ray bundle 8 from theoptical spot, the aperture stop 35 sets the outer diameter of the raybundle to a predetermined value and thereafter the laser beam passesonly through the peripheral portion of objective lens 14 to go outthereof. It is then irradiated onto the substrate 38 in the form of aBessel beam as an optical spot converged up to the diffraction limit.The Bessel beam has a diffraction beam intensity distribution composedof a very thin linear center beam having a large intensity in thedirection of the optical axis and numerous concentric cylindricalhigher-order diffraction beams present around the center beam.

Scanning speeds and scanning timings in synchronization with each otherare set for the X-Y scanner 12 and X-Y stage 36 based on a controlsignal output from the control unit 38. Then the two-dimensionalscanning, for example the raster scanning, with the Bessel beam iscarried out on the surface of substrate 38 through the X-Y scanner 12and X-Y stage 36. On the photosensitive agent 39 exposure is effectedonly in a portion irradiated by the Bessel beam having an opticalintensity portion exceeding a certain threshold. Accordingly, variouspatterns of integrated circuits input in the control unit 37 can beprinted on the photosensitive agent 39 on the surface of substrate 38.

In such an embodiment of optical writing apparatus as described above,the laser beam passes as the cylindrical ray bundle only through theperipheral portion of the objective lens, whereby the Bessel beam withthe thin linear center beam formed to have a large intensity in thedirection of the optical axis is irradiated onto the surface ofsubstrate. Thus, the Bessel beam has a long focal depth, so thatexposure can be effected on the photosensitive agent on the substrateirrespective of unevenness of the substrate surface. This can obviateprecise alignment of the position of optical spot of the laser beam withthe photosensitive agent, improving the efficiency of operation.

The Bessel beam includes the concentric cylindrical peripheral beamsaround the linear center beam, but they will cause no problem if writinglight for the photosensitive agent having a high contrast has only twotypes of levels of on/off. Thus, the Bessel beam has a high resolutionand therefore, with exposure of a pattern of integrated circuits on thephotosensitive agent on the substrate, the degree of integration can beincreased for the integrated circuits formed based on this pastern.

FIG. 12 is a structural drawing to show an embodiment associated with alaser repair apparatus using the laser scanning optical system of thepresent invention. The present embodiment is constructed almost in thesame manner as the above embodiment associated with the optical writingapparatus. However, an IC (Integrated Circuits) chip 42 is set insteadof the substrate 38 on the X-Y stage 36. Also, an angular deflectionprism 40 is arranged to be interposed by unrepresented moving means inthe optical path between the imaging lens 13 and the objective lens 14.In this case, an eyepiece 41 is set along a direction in which areflection beam from the IC chip 42 is outgoing through the objectivelens 14 and the aperture stop 35 from the deflection prism 40.

The deflection prism 40 has a total reflection surface for angulardeflection in the direction of 30° or 45° with respect to the opticalaxis, which reflects light incident thereinto from the IC chip 42through the objective lens 14 and the aperture stop 35. The eyepiece 41converges the light incident thereinto from the deflection prism 40 infocus. The IC chip 42 includes integrated circuits on the surface regionand is located on the rear image plane of objective lens 14. Theintegrated circuits in the IC chip 42 include a local defect having beenmade during fabrication or having occurred in operation.

Next described is the operation of the above embodiment associated withthe laser repair apparatus.

The present embodiment is operated almost in the same manner as theabove embodiment associated with the optical writing apparatus. However,the outgoing laser beam 2 having passed only through the peripheralportion of objective lens 14 is irradiated onto the IC chip 42 in theform of a Bessel beam as an optical spot converged up to the diffractionlimit. The IC chip 42 is etched only in a portion irradiated by theBessel beam having an optical intensity portion exceeding apredetermined threshold. Then the integrated circuits formed in the ICchip 42 are repaired matching with the various patterns of integratedcircuits input in the control unit 37.

In such an embodiment associated with the laser repair apparatus asdescribed above, the laser beam passes as the cylindrical ray bundleonly through the peripheral portion of objective lens, whereby theBessel beam with a thin linear center beam formed with a large intensityin the direction of the optical axis is irradiated onto the surface ofsubstrate. Thus, the Bessel beam has a long focal depth, so that theintegrated circuits in the surface region of IC chip can be etched freeof influence of unevenness of the integrated circuits themselves. Thiscan obviate precise alignment of the optical spot of laser beam withrespect to the substrate of laser beam, improving the efficiency ofoperation.

The Bessel beam includes the concentric cylindrical peripheral beamsaround the linear center beam, but the energy of the peripheral beamscan be reduced relative to the center beam by adjustment of laser power.The Bessel beam thus has a high resolution, so that a structurally finedefect having occurred in the integrated circuits can be repaired.

The present invention is by no means limited to the above specificembodiments, but can have various modifications.

For example, the above embodiments associated with the fluorescencemicroscope were so arranged that the dichroic mirror was located in theoptical path between the beam compressor and the X-Y scanner. However,the dichroic mirror can be set at any position as long as it is in theoptical path between the laser and the X-Y scanner, obtaining the sameoperational effect as the above embodiments.

Also, the above third embodiment associated with the fluorescencemicroscope employed three dichroic mirrors. However, any number otherthan three, of dichroic mirrors may be set as long as the number ofdichroic mirrors corresponds to the number of types of fluorescent dyesapplied to the sample, obtaining the same operational effect as in theabove embodiments.

As detailed above, the optical converting unit is composed of two axiconprisms in the laser scanning optical system of the present invention.These axicon prisms are so arranged that the apexes thereof are opposedforward or backward to each other at a predetermined distance and theoptical axes thereof are coincident with each other, and are made ofrespective materials having the same index of refraction and shaped witha same apical angle. By this, the laser beam incident as a beam ofparallel rays into the optical converting unit has an annularcross-sectional intensity perpendicular to the optical axis and becomesa cylindrical ray bundle as a beam of parallel rays traveling in thedirection parallel to the optical axis. Therefore, the laser beamoutgoing from the optical converting unit is changed in the travelingdirection by the optical scanning unit to be scanned and is thenconverged by the optical converging unit to become a Bessel beam.

Accordingly, the cylindrical ray bundle is formed without a loss inintensity of laser beam, so that the invention can provide a laserscanning optical system which can perform scanning of the Bessel beamhaving a high energy utilization factor, a high resolution and a longfocal depth.

On the other hand, the laser scanning optical apparatus of the presentinvention is so arranged that a laser beam emitted from the light sourceis irradiated onto a certain sample through the laser scanning opticalsystem of the present invention. By this, the sample is scanned with theBessel beam having a high energy density, a high resolution and a longfocal depth.

Here, in the case that the light source oscillates a laser beam of veryshort time duration pulse, that the sample is labeled with apredetermined fluorescent dye, and that the apparatus is furtherequipped with an optical detecting unit for detecting fluorescenceemitted from the sample based on the multiphoton absorption withirradiation of the Bessel beam, the fluorescence appears only from aposition irradiated by the center beam in the Bessel beam based on poweradjustment of laser beam, which can prevent false signals from beingproduced by the higher-order diffraction beams in the Bessel beam. Thiscan improve a resolution of a microscopic image of the sample asobtained by storing output signals from the optical detecting unit aspixel data in synchronization with scanning of laser beam. Also, basedon two-dimensional scanning of the Bessel beam, a microscopic image astwo-dimensional projection of a three-dimensional image of the sample isobtained as integral values in the thickness direction, which requiresno three-dimensional scanning inside the sample and greatly reduces thescanning time of optical spot as compared with the conventionalapparatus. In the case that the optical detecting unit is composed offirst and second photoelectric detectors for detecting fluorescenceemitted from the surface side and from the back side, respectively, ofthe sample, the fluorescence emitted from the sample reaches thephotoelectric detectors with almost no loss, which can enhance thecontrast in the microscopic image produced based on addition of outputsignals from the photoelectric detectors. Therefore, a low-power lasercan be used as a light source for emitting the laser beam, which canreduce a damage on an organism sample or another sample.

In the case that the surface of sample is coated with a predeterminedphotosensitive agent and that a predetermined pattern is formed on thephotosensitive agent based on exposure with irradiation of the Besselbeam, exposure on the photosensitive agent on the substrate is effectedfree of influence of unevenness of substrate surface with the Besselbeam having a long focal depth, which obviates precise alignment of theposition of optical spot of laser beam with respect to thephotosensitive agent, improving the efficiency of operation. When apattern of integrated circuits is printed on the photosensitive agent onthe substrate with the Bessel beam having a high resolution, the degreeof integration can be increased for the integrated circuits formed basedon the pattern.

Further, in the case that the surface of sample is exposed to theoutside and that the surface region of sample is shaped in apredetermined shape based on excitation with irradiation of the Besselbeam, the surface region of substrate can be etched free of theinfluence of unevenness of itself by the Bessel beam having a long focaldepth, which can obviate precise alignment of the position of opticalspot of laser beam with respect to the substrate, improving theefficiency of operation. If the sample is an IC chip having integratedcircuits in the surface region, a structurally fine defect occurring inthe integrated circuits can be repaired with the Bessel beam having ahigh resolution.

Accordingly, performing scanning of the Bessel beam having a high energyutilization factor, a high resolution and a long focal depth, theinvention can provide a laser scanning optical system as used as afluorescence microscope, an optical writing apparatus, an integratedcircuit repair apparatus, etc.

What is claimed is:
 1. A fluorescence microscope comprising:a laserscanning unit including:a stage for supporting a fluorescence samplethereon; a laser beam generator; and a beam scanning unit for causing alaser beam from said laser beam generator to spot scan a sample to causefluorescence generated by multiphoton absorption; a first detector fordetecting fluorescence emitted from a laser beam incident side of saidsample to output a first signal corresponding to the detectedfluorescence; a second detector for detecting fluorescence emitted froma laser beam transmitted side of the sample to output a second signalcorresponding to the detected fluorescence; means for summing andamplifying the first and second signals; and means for displaying amicroscopic image of the sample in synchronization with scanning of thelaser beam based on the amplified summed signals.
 2. A fluorescencemicroscope according to claim 1,wherein said laser beam generatorcomprises an optical converting unit for shaping a laser beam incidentthereinto as a beam of parallel rays into a cylindrical ray bundlethereof; and wherein said beam scanning unit comprises:an opticalscanning part for changing a traveling direction of said laser beamincident from said optical converting unit thereinto to scan; and anoptical converging part for converging said laser beam incident fromsaid optical scanning part thereinto to produce a Bessel beam, saidoptical converting part comprising two axicon prisms which are arrangedsuch that apexes thereof are opposed forward or backward to each otherat a predetermined distance and optical axes thereof are coincident witheach other and which are made of respective materials having a samerefractive index and shaped with a same apical angle.
 3. A fluorescencemicroscope according to claim 2, wherein said optical converting parthas moving means for variably setting a distance between said two axiconprisms.
 4. A fluorescence microscope according to claim 2, furthercomprising a beam expander disposed either on the entrance side or onthe exit side of said optical converting part, for expanding a diameterof the ray bundle of said laser beam.
 5. A fluorescence microscopeaccording to claim 2, further comprising a beam reducer disposed eitheron the entrance side or on the exit side of said optical convertingunit, for reducing a diameter of the ray bundle of said laser beam.
 6. Afluorescence microscope according to claim 1, wherein said laser beamgenerator provides a pulsed laser beam, the pulses having a very shorttime duration, and wherein said sample is labeled with a predeterminedfluorescence dye.